A basic medium commonly used for capturing and sustaining the growth of plant embryogenic tissue is known as the Murashige Skoog (MS) medium. This medium is based on the ratios and concentrations of minerals found in tobacco leaves. It has been possible to successfully grow embryogenic tissue from many species in media which have slight variations from the concentrations/ratio of the minerals in the MS medium. Unfortunately, the MS medium or variations are not ideal for growing coniferous tissue.
One growth medium known as the Weyerhaeuser medium (Gupta and Pullman 1990, 1991a, 1991b) has been developed particularly for coniferous embryogenic tissue, based on chemical analysis of the composition of pine seeds. This medium is significantly different from the MS medium in both its ratios and concentrations. Unfortunately the Weyerhaeuser medium only works in relation to a few conifer genotypes and therefore is too specific for general conifer propagation. There are other problems associated with the Weyerhaeuser medium and the media referred to above which will become apparent from the following discussions.
With embryogenesis, an aim is to obtain as many embryos as possible from a single seed. The natural growth of a seed proceeds in six main stages:
a) The first stage has an embryo consisting of between one to three cells attached to the archegonium and positioned within the corrosion cavity of the seed. PA1 b) The second stage has a number of embryos multiplying and developing with each embryo having less than 64 cells. It is usually at this stage (zygotic polyembryogenesis) that embryos are placed onto or into the growth medium. PA1 c) The third stage is growth of the embryo away from the archegonium and towards the end of the corrosion cavity. The long axis of the embryo develops and assumes a cylindrical shape with a complex of elongated cells, the suspensors, at one end, and with a rounded head at the other end where the apical meristem will eventualy develop. This is known as a bullet stage. PA1 d) The fourth stage is the development of cotyledonary tissue at the apex of the embryo, at the root end of which is a group of cells known as the suspensor zone. PA1 e) The fifth stage is the further development and maturation of embryos, with the formation and greening of cotyledons, formation of an epicotyl or shoot apex, and formation of a hypocotyl. This stage of development ends with emergence of a root (radicle), that is, the process of germination. PA1 f) The sixth stage is the establishment of the germinated embryo as a plant capable of growth in soil.
Prior growth media do not encourage the natural zygotic polyembryogenic state which occurs in the seeds. Instead, with the previous media it has necessary to dissect out embryos at the cotyledonary stage. Plant growth regulators (hormones) are then applied to the cells of the body of the embryo or at the point of attachment of the suspensor to encourage the cells to differentiate back to nonspecialized cells which can then be multiplied. This process, often referred to in the literature as somatic embryogenesis, has been widely reported for Picea (Spruce) species, and to a lesser degree for other conifers.
One problem with the above process is that there is in effect double handling involving first the development of specialised tissue, reversion of same to basic cell types and then re-growth of the tissue to form mature embryos. Another problem is that the growth hormones used (such as 2,4-D) may induce somaclonal variation. That is, the ideal genotype which is being cloned may be corrupted by the growth hormones and the resultant embryo may not be true to type.
It is an object of the present invention to address the above problems, or at least to provide the public with a useful choice.
Further objects and advantages of the present invention will become apparent from the following-description.